The impaired function of abnormal variants of Factor IX could result from (1) failure to undergo activation, or (2) lack of biologic activity despite normal activation. The latter could stem from structural alterations that interfere either with the function of the activity site or with the interaction of the enzyme with its cofactors, CA++, phospholipid and Factor VIIIa. We will study the kinetics of activation of two purified variants utilizing radiolabelled proteins and monitoring the radioactivity (125I) profiles on SDS gels and by 3H-activation peptide release assay after attempted activation with Factor XIa, tissue factor-Factor VII, and RVV. We will examine the availability of active site in activated molecules by their ability (a) to interact with AT-III, (b) to activate Factor VII, and (c) to activate Factor X in the absence of cofactors. The ability of activated variants to activate Factor X will be assessed from the kinetics of release of 3H-activation peptide from 3H-Factor X, in the presence and absence of cofactors. Direct binding of metal ions to these variants will be studied by equilibrium dialysis (85Sr++, 45CA++) a specific CA++ electrode (CA++), EPR method (Mn++) and fluorescence enhancement (Tb+++). We will examine phospholipid interactions by fluorescence energy transfer and fluorescence light scattering techniques. All of the above findings will be contrasted with findings obtained with purified normal Factor IX. The structural alterations in the abnormal molecules could stem from either deletion or substitution of an amino acid(s) or from abnormal postribosomal modifications. We will characterize the two variants with respect to mol. wt., amino terminal amino acid residues (and/or sequence), amino acid composition, Gama-carboxylutamic acid residues, isoelectric focusing point, neutral sugar and sialic acid content. Isoelectric focusing will also be performed on decarboxylated and disialylated normal and abnormal variants of Factor IX to determine the composite charge differences in their polypeptide backbones. Relationship of structural alterations to the functional impairment in varient molecules will contribute to our understanding on a molecular basis of how normal Factor IX participates in clotting and why abnormal variants fail to function in hemostasis.